The sodium sulfate of the delta(8,9) derivative of estrone [delta(8,9)DHES] is present in minor amounts of about 3-4% in natural conjugated estrogen compositions, for instance in the commercially available product Premarin.RTM. which is being used in hormone replacement therapy.
In addition to estrone sodium sulfate, several components have been identified in natural conjugated estrogen compositions among others the sodium sulfates of equilin (in amounts of 22.5-30.5%), 17 alfa dihydro equilin (13.3-19.5%), 17 beta dihydro equilin (0.5-4.0%), 17 alfa estradiol (2.5-9.5%) 17 beta estradiol (&lt;4.5%) and delta(8,9)-dehydro estrone (&lt;12.5%) (U.S. Pharmacopoeia, 1995, p. 627).
It has been suggested in SCRIP no. 2049 (1995), p. 15 that minor amounts of delta(8,9)DHES could have a significant contribution to the effect of conjugated estrogens. It has further been suggested that delta(8,9)DHES, which has a relatively low affinity to the estrogen receptor, has a high functional activity, which may play a role in the reported LDL-cholesterol-reducing properties and cardiovascular effects of conjugated estrogens, in particular of Premarin.RTM.. Data reveal that delta(8,9)DHES contributes to about 18% of Premarin's circulating estrogens. It is therefore of importance to obtain an easy method of production of sulfated mixtures of delta(8,9)DHE.
Apart from cumbersome total synthesis, J. C. Jacquesy et al., Chem. Abstr. 76 (1972), 154000f disclosed isomerization of equilin in hyperacidic media. Conversion to delta(8,9)DHE was achieved by using hydrogen fluoride or hydrogen fluoride/antimony fluoride at -30.degree. C. It is evident that such dangerous reaction conditions are completely unsuitable and unacceptable for large scale production of delta(8,9)DHE. Moreover, in U.S. Pat. No. 5,395,831, wherein the method of Jacquesy is applied, it has been disclosed that said hydrogen fluoride method does not provide pure delta(8,9)DHE, but in addition thereto 10% of the unwanted delta(9,11)-isomer. Methods of production which are commercially acceptable, whether or not through isomerization of equilin, thus have not been disclosed.
Synthesis of sulfated esters of steroids has been described in the literature. As summarized by Jenkins and Sandberg (Methods in Enzymology, 15, 351-358, 1969) one of the methods involves the sulfatation of mono- and dihydroxy C-18 and C-19 steroid compounds utilizing pyridine sulfuric acid complexes. This process, however, has been applied to individual, i.e. free from other, steroid compounds. The process according to the present invention has the advantage that in a single reaction mixture sulfated estrogens can be obtained in a specific ratio. It has surprisingly been found that although the estrogens present in the natural conjugated estrogen preparations have different physical properties, such as crystallization behavior and solubility, the ratio of sulfated products in the sulfatation reaction mixture reflects the amounts of the input components. Therefore, a one pot reaction suffices to prepare the sulfated estrogen mixture. In addition, this reaction can, when appropriate, be coupled directly to the isomerization reaction by which delta(8,9)DHE is prepared, i.e. isomerization of equilin or a derivative thereof to said derivative. In this reaction equilin or a derivative thereof is treated with a lithium salt of ethylenediamine or with lithium amide in dimethylsulfoxide.